Fig. 6.

K8(S431) phosphorylation is sufficient to trigger perinuclear keratin organization in Panc-1 cells. (A–C) Panc-1 cells were transfected with K8(SE),K18(WT) (A), K8(SA),K18(WT) (B) or K8(SE),K18(SA) (C) and treated with 15 μM SPC and/or 10 μM U0126 as indicated (eCFP-tagged K8; eYFP-tagged K18). Keratin was detected within the 488 channel. Images show representative cells. Graphs display quantification of cells with perinuclear or ramified keratin compared with the total number of transfected cells (means ± s.e.m.). (D) Panc-1 cells were transfected with either eCFP–K8(WT) or eYFP–K8(SE) and stained with pan-CK antibody, followed by labelling with Alexa-Fluor-647 antibody (Pan-CK). Images were taken using a confocal microscope and keratin was detected within two channels. (E) Quantification of cytokeratin organization. Cytokeratin organization was quantified in cells expressing wild-type K8–CFP or K8(SE)–CFP mutant. Images represent ortho-max projections of confocal image sections with three linear ROIs in the perinuclear and the cytoplasmic region, respectively. Left graph shows intensity ratio of perinuclear to cytoplasmic ROIs. Cells overexpressing the phosphomimetic K8 mutant (CFP–K8 se) exhibit an increase in fluorescence intensity in the perinuclear area compared with cells overexpressing wild-type K8, demonstrating a marked difference in cytokeratin redistribution upon phosphorylation that can be quantified. Right graph shows height of cells (Z-volume) calculated from confocal image stacks. **P<0.01; ***P<0.001.